Physiology and Biochemistry 144 (2019) 445–454

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Plant Physiology and Biochemistry

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Research article Iron nano-complexes and iron chelate improve biological activities of sweet (Ocimum basilicum L.) T

∗ Vahid Tavallalia, , Mahmoud Kianib, Shadi Hojatia a Department of Agriculture, Payame Noor University (PNU), Tehran, Iran b Faculty of Medicinal , Amol University of Special Modern Technologies (AUSMT), Amol, Iran

ARTICLE INFO ABSTRACT

Keywords: In this study, antioxidant and antimicrobial activities of basil (Ocimum basilicum L.) essential oil (EO) in response Antimicrobial activity to different Fe sources (Fe-arginine, Fe-glycine, and Fe-histidine nano-complexes and Fe-EDDHA) were ex- Antioxidant activity amined. EO samples were predominantly constituted by the phenylpropanoid methyl chavicol (53–89.5%). Sweet basil Application of Fe nano-complexes significantly increased the occurrence and concentration of sesquiterpenes, Essential oil while decreased the content of oxygenated monoterpenes. Antioxidant activity of basil EOs was evaluated using Nanoparticle free radical 2,2-diphenyl-1-picrylhydrazyl, Nitric oxide, H O and Thiobarbituric acid reactive substances Ocimum basilicum 2 2 scavenging assays, and in all assays the highest and the lowest activities were recorded in supplied with Fe- histidine nano-complex (1.02, 1.62, 2.21, 3.22 mg mL-1) and control (3.89, 4.89, 5.52, 6.79 mg mL-1), re- spectively. Fe-histidine nano-complex was the most effective treatment to inhibit fungal (C. albicans: 0.058 mg mL-1; A. niger: 0.066 mg mL-1), Gram-negative (E. coli: 0.181 mg mL-1; S. typhimurium: 0.163 mg mL-1) and Gram-positive (B. subtilis: 0.033 mg mL-1; S. aureus: 0.002 mg mL-1) growth. In conclusion, application of iron nano-complexes significantly altered biological and pharmacological characteristics of basil EOs. Our results are quite encouraging since EOs exhibited potent antioxidant effect and antimicrobial activities.

1. Introduction Iron (Fe) is an essential micronutrient for plants and plays a key role in regulating numerous cellular processes, including chlorophyll bio- Basil (Ocimum basilicum L.) is an annual in the family synthesis, photosynthesis, and mitochondrial respiration (Ghasemi Lamiaceae, native to India, Africa, and southern Asia, and is commer- et al., 2014). While Fe is abundant in soil, the available Fe in soil for cially cultivated in different parts of the world. Basil is a popular cu- plants is often insufficient due to low iron solubility. Iron deficiency is linary herb, and its essential oils (EOs) have been used extensively for one of the most important factors limiting crop production in the world. many years in the flavoring of food products, perfumery, and dental and Fe deficiency-induced chlorosis is a major nutritional disorder in crops oral products. Basil EOs (BEOs) and their principal constituents have growing in alkaline and calcareous soils. A large proportion of the total recently been used for increasing the shelf life of food products due to land area of Iran is dominated by highly calcareous soils (Ziaeian and their antimicrobial activity against a wide range of bacteria, yeast, and Malakouti, 2001). Application of synthetic iron chelates is effective in mold (Suppakul et al., 2003). Additionally, drugs derived from basil counteracting Fe deficiency symptoms of plants grown on calcareous have been consumed traditionally for the treatment of various disorders soils and is the most commonly applied technique in agriculture (Vadas and diseases such as warts, inflammations, colds, and headaches. et al., 2007). However, because of some serious drawbacks e.g. cost, Ocimum basilicum is characterized by a great variability in its che- environmental side effects, susceptibility to photodegradation, their motypes, with the major compounds being linalool, eugenol, methyl application is not regarded as a sustainable practice (Metsarinne et al., chavicol, methyl eugenol, geraniol, geranial and neral, methyl cinna- 2004; Vadas et al., 2007; Souri and Hatamian, 2019). mate, which is to a large extent genotype-specific(Grayer et al., 1996; Nanomaterials are used in practically every aspect of modern life. Burducea et al., 2018; Zheljazkov et al., 2008). In addition to genetic Nanoparticles (NPs) display unique size-dependent optical, physico- makeup, extrinsic factors such as environmental conditions and agro- chemical, and biological properties that are extremely looked-for in nomic practices (nutrient management) have also been evidenced to many disciplines including agriculture, medicine, environment, etc. play a part. (Hao et al., 2019). Nowadays, it is widely believed that NPs should be

∗ Corresponding author. E-mail address: [email protected] (V. Tavallali). https://doi.org/10.1016/j.plaphy.2019.10.021 Received 8 August 2019; Received in revised form 15 October 2019; Accepted 16 October 2019 Available online 18 October 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved. V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454 used in the field of soil–plant nutrition to achieve sustainable devel- opment of agricultural production with minimal environmental impacts (Rui et al., 2016). Nano-complexes increase crop production via en- hancing a series of physiological processes including seed germination rate, photosynthetic activity, seedling growth, protein and carbohy- drate synthesis, and nitrogen metabolism (Huang et al., 2015; Rui et al., 2016). Nano-fertilizers have also exhibited promise for increasing nu- trient use efficiency, declining nutrients deprivation, and reducing salinity stress in plants (Huang et al., 2015; Zia-ur-Rehman et al., 2018). Practically, the majority of Iranian farmers in arid and semi-arid climates use different types of Fe chelates; however, the application of these fertilizers still remains questionable. NPs are expected to be the ideal candidates for use in these regions. The effects of chelated Fe on growth and development of medicinal plants have been extensively studied. However, plant responses to Fe-nano complexes remain poorly understood. To bridge this information gap, we examined the effec- tiveness of foliar application of iron-amino acid nano-complexes in comparison with a commercial chelated Fe on some biological activities of BEOs. This was an attempt to generate new information on the ef- ficacy of nanoscale iron fertilizers on the growth and development of basil, and to develop a technical approach for the agricultural appli- cation of nanomaterials. It is to be noted that the increased efficiency of a product may encourage the farmers to use the product more profit- Fig. 1. A sample of Fe-Histidine (n[Fe(His)3]) nano complex used in this study. ably. 2.3. EO extraction 2. Materials and methods The plant samples were shaded at ambient temperature, and then 2.1. Plant material and treatments 50 g of each sample was hydro-distilled using a Clevenger-type appa- ratus for 3 h (British Pharmacopoeia, 1988). EO samples were dehy- The experiment was conducted at the greenhouse of Shiraz Payame drated over anhydrous sodium sulfate and stored at 4 °C until analysis. −1 Noor University through spring 2018 (Shiraz, Iran). The soil texture The BEO yields were calculated based on g oil content 100 g dried was characterized as sandy loamy (Alloway, 2004). Other chemical and herb. physical characteristics of the soil are as follows: − − ECe (1.3 dS m 1), pH (7.1), organic (8.8 g kg 1), CEC (11 Cmc 2.4. GC & GC/MS analysis − − − kg 1), K (61 mg kg 1), P (12 mg kg 1), N (0.08%) and Zn − − (1.5 mg kg 1) and Fe (1.01 mg kg 1). Gas chromatography (GC) and Gas chromatography-mass spectro- −1 50 mg N and P kg soil (as NH4NO3 and KH2PO4), and 5 mg Cu, Zn metry (GC-MS) analyses were performed using an autosampler Agilent −1 and Mn kg soil (as CuSO4.5H2O, ZnSO4.7H2O, and MnSO4.H2O) were 7683B, fitted with a flame ionization detector (FID), HP-5 fused silica applied to the soil, respectively. Seven-liter plastic pots were used, and column (30 m × 0.32 mm i.d. × 0.25 μm). GC/MS analysis was per- for each pot, 6 kg of soil was weighed. In each pot, 20 selected Ocimum formed using an Agilent gas chromatograph fitted with 5975-C mass basilicum L. seeds purchased from an authenticated company (Shiraz, spectrometer joint with a capillary HP-5MS column (30 m × 0.25 mm Iran) were sown. Pots were irrigated twice a week with deionized i.d. × 0.25 μm). The carrier gas was Helium at the ionization voltage of water. After 15 days, in each pot, the plants were thinned to ten steady 70 eV. Oven temperature program was as follows: 60 °C for 3 min, then − and similar stands. Each treatment included 8 pots containing 10 an increase of 3 °C min 1 until 150 °C, afterward, a further increase of − seedlings in each pot. Since thinning and prior to the flowering incep- 3 °C min 1 until 260 °C, and this temperature was maintained for tion, the following treatments were foliarly applied: Treatment 1 (T1): 3 min. The injector and detector temperatures were set to 230 and

Fe-Arginine (n[Fe(Arg)3]), Treatment 2 (T2): Fe-Glycine (n[Fe(Gly)3]), 250 °C, respectively. Identification of oil components was made by Treatment 3 (T3): Fe-Histidine (n[Fe(His)3]), and Treatment 4 (T4): Fe- comparison of their mass spectra and retention indices with those of the EDDHA. One liter of each Fe source (0.2% (w/v)) was applied. mass spectral reference library and with those given in the literature Deionized water was sprayed as control. Plants were collected at full (Adams, 2007). The retention indices were determined in relation to a bloom stage. The nano-complex fertilizers were obtained from Zist Nano homologous series of n-alkanes (C8–C24) under the same operating Fanavarn Atiye Pajooh (Shiraz, Iran). All Fe-amino acid nano complexes conditions. Component relative percentages were calculated based on were visually similar and were produced as yellow-orange fine powders GC peak areas without using correction factors. (Fig. 1). 2.5. Antioxidant activity 2.2. Instrumentation and characterization Four antioxidant assays i.e. free radical 2,2-diphenyl-1-picrylhy- The following techniques were used to verify the successful bio- drazyl (DPPH), Nitric oxide (NO), hydrogen peroxide (H2O2) and synthesis of Fe-amino acid nano-complexes. The size of the nano- Thiobarbituric acid reactive substances (TBARS) scavenging assays complexes was observed using a transmission electron microscope were used for determining the antioxidant capacity of BEOs. (TEM) (100 kV Philips, EM208). The energy dispersive X-ray (EDX) spectroscopy analysis (Tescan Vega II, with a Rontec detector) was used 2.5.1. DPPH assay to confirm the presence of pure elemental Fe. FT-IR analysis was carried The antioxidant activity of EO samples was determined by free ra- − out using a Tensor II FT-IR spectrometer set to 500–4000 cm 1. dical (DPPH) scavenging assay method, as previously described (Burits

446 V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454 et al., 2001). DPPH radical is a stable organic free radical with an ab- scavenging activity assay is widely used to measure antioxidant activity sorption band at 517 nm. It loses this absorption when accepting an in food and physiological systems. TBARS scavenging activities of BEOs electron or a free radical species, which results in a visually noticeable were carried out according to the previously described method pro- discoloration from purple to yellow. It can accommodate many samples posed by Kavoosi and Rowshan (2013). Briefly, 0.2 mL of the EOs − in a short period and is sensitive enough to detect active ingredients at (0–0.4 mg mL 1 in DMSO) was mixed with MDA (0.1 mM in acetic acid, low concentrations (Do et al., 2014). In short, 20 μL of EOs (0–0.4 mg pH = 4). Solutions were kept at 36 °C for 120 min. After adding one mL-1 in DMSO) was mixed with 210 μL of 125 mmol L-1 radical dilution volume of thiobarbituric acid (0.3 mM in acetic acid pH = 4), the so- of DPPH in methanol at room temperature for 30 min using an absor- lutions were incubated at 90 °C for 60 min. Then, the mixtures were bance microplate reader (EL × 808, BioTek, USA) at 515 nm wave- chilled at ambient temperature. Then, the absorbance of solutions was length, the absorbance of solutions was assessed. The values of samples recorded at 532 nm using a spectrophotometer (Shimadzu 1601, providing 50% inhibition to restrain DPPH radical formation (IC50) Japan). The following equation was used to measure the percentage of were assigned by the nonlinear regression plots using MATLAB TBARS scavenging activity: (MathWorks®, USA). The percentage of ROS scavenging was assessed [(A532 ˗ A532 )/A532 ] based on the following formula, where Asample and Ablank correspond blank sample blank to the absorption values of the test solution and the blank solution, respectively. For control and blank, DPPH (without plant extract) and 2.6. Antifungal and antibacterial assays methanol were used: Bacteria and fungi were collected from the Persian Type Culture [(A515sample‒A515blank)∕A515control] × 100 Collection (PTCC), Tehran, Iran. Two foodborne gram-positive bacteria [Bacillus subtilis PTCC 1023 (ATCC 6633) and Staphylococcus aureus 2.5.2. NO radical scavenging assay PTCC 1112 (ATCC 6538)], two foodborne Gram-negative bacteria Reactive nitrogen species (RNS) are a family of free radicals derived [Escherichia coli PTCC 1330 (ATCC 8739) and Salmonella typhimurium from the interaction of nitric oxide (NO) with oxygen or reactive PTCC 1609 (Iran isolate) and two foodborne fungi [Candida albicans oxygen species. NO is classified as a free radical because of its unpaired PTCC 5027 (ATCC 10231) and Aspergillus niger PTCC 5010 (ATCC electron and displays important reactivity with certain types of proteins 9142)] were tested. The minimum inhibitory concentration (MIC) of − and other free radicals such as superoxide. Generally, in vitro quenching serial dilutions of EOs (0–0.3 mg mL 1) against the pathogens was of NO radical is one of the methods that can be used to determine an- measured according to the microdilution procedure proposed by tioxidant activity in food and physiological systems. The procedure is Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2012). Sus- based on the principle that sodium nitroprusside in aqueous solution at pended fungi and bacteria strains in Luria–Bertani media were tuned to physiological pH spontaneously generates nitric oxide which interacts 0.6 McFarland standards at 640 nm (108 CFU/mL). Densities were di- with oxygen to produce nitrite ions that can be estimated using Griess luted to 105 (CFU/ml) with Luria–Bertani. The EOs (0.5 mL) and sus- reagent. Scavengers of nitric oxide compete with oxygen, leading to pensions of fungi and bacteria (0.5 mL) were added to 1.5 mL micro- reduced production of nitrite ions (Boora et al., 2014). tubes. The mixtures were then shaken with incubating at 36 °C for 24 h. NO radical scavenging activity of EOs were assessed following the In control (blank), EOs were excluded from the medium containing previously described method by Kavoosi and Rowshan (2013). In short, bacteria and fungi. Sterile control was a medium without fungi and − 0.2 mL of the EOs (0–0.4 mg mL 1 in DMSO) was mixed with 0.5 mL of bacteria. Positive control contained Ketoconazole, Ampicillin and −1 −1 NaNO2 (0.02 mg mL in 100 mM Na3C6H5O7 (sodium citrate)). Then, Gentamicin (Pourateb, Iran, 0.015 mg mL ) for fungi, Gram-positive at 36 °C, the combination was kept for 120 min. Then, 0.6 mL of Griess and Gram-negative bacteria, respectively. To show the growth inhibi- reagent was added to the mixture. The absorbance of solutions was tion, absorbance (A) of the culture medium was measured using a measured at 540 nm using a spectrophotometer (Shimadzu 1601, spectrophotometer (Shimadzu 1601, Japan). The following equation Japan). The following equation was employed to obtain the percentage was employed to obtain the percentage of growth inhibition: of RNS scavenging: [(A640blank - A640sample)/A640blank] × 100 [(A540blank ˗ A540sample)/A540blank] It should be noted that during incubation, EO samples caused dis- coloration of the culture media, which may result in an inaccurate es-

2.5.3. H2O2 scavenging assay timation of percent inhibition. This discoloration was accounted for by Hydrogen peroxide (H2O2) can be formed in vivo by many enzymes incubating EOs in uninoculated media for 24 h, measuring the change such as superoxide dismutase. It can cross membranes and may slowly in turbidity, and adding this correction factor to final OD values oxidize a wide range of compounds (Gülçin et al., 2007). H2O2 (Donaldson et al., 2005). scavenging activity of BEOs was assessed following the method pro- posed by Kavoosi and Rowshan (2013). In detail, 0.2 mL of EOs 2.7. Statistical analysis −1 (0–0.4 mg mL in DMSO) was blended with 2.0 mL of H2O2 (100 mM in 200 mM phosphate buffer, pH = 7.4). The mixtures were then in- All experiments were carried out in eight replications (eight pots), cubated at 36 °C for 75 min. The absorbance (A) of solutions was re- and data were analyzed using analysis of variance (ANOVA), and sig- corded at 230 nm using a spectrophotometer (Shimadzu 1601, Japan). nificant differences among means at (p < 0.05) were determined by A solution containing phosphate buffer without H2O2 was used as Duncan's multiple range test using SPSS 20 (SPSS Inc., USA) software. blank. The following equation was used to obtain the percentage of H2O2 scavenging of each sample: 3. Results and discussion

[(A230blank ˗ A230sample)/A230blank] 3.1. Synthesis of Fe-amino acid nano complexes

2.5.4. TBARS scavenging assay 3.1.1. TEM The thiobarbituric acid reactive substances (TBARS) are formed as a The particle size and shape of biosynthesized Fe amino acid nano byproduct of lipid peroxidation, which can be detected by the TBARS complexes were analyzed by using TEM technology (Fig. 2). The TEM assay using thiobarbituric acid as a reagent (Ghani et al., 2017). TBARS images of nano-complexes revealed that they are in the nanoscale range

447 V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454

Fig. 2. Transmission electron microscopy images of (a) Fe-Histidine (n[Fe(His)3]), (b) Fe-Glycine (n[Fe(Gly)3]) and (c) Fe-Arginine (n[Fe(Arg)3]) nano-complexes.

(~5–20 nm) and have a relatively uniform distribution with spherical quantitative differences were observed (Table 2). The minor and major shapes. EO components were variable in occurrence and concentration among the tested samples. In total, 72 compounds were identified accounting 3.1.2. EDX for over 99% of the total oils. A number of compounds belonging to Energy-dispersive X-ray spectroscopy (EDX) is an analytical tech- different chemical classes have been reported, among which phenyl- nique used for the elemental analysis or chemical characterization of a propanoids have been shown to play a significant role in the estab- sample. Results of EDX analysis confirmed the presence of Fe NPs, along lishment of chemotypes in O. basilicum: (a) linalool, (b) methyl cha- with three other elements including C, N, and O (Fig. 3). vicol, (c) linalool-methyl chavicol, (d) linalool-eugenol, and (e) methyl chavicol-methyl eugenol chemotypes are common ones. Although 3.1.3. FT-IR phenylpropanoids are not common in EO-bearing plants, O. basilicum Fourier Transform-Infrared Spectroscopy (FT-IR) is an analytical EO commonly contains abundant proportions of these compounds. In technique used to identify organic (and in some cases inorganic) ma- the current study, all of the EO samples (notably control) were pre- terials. We performed an FT-IR analysis of free amino acids (histidine, dominantly constituted by the phenylpropanoid methyl chavicol, ran- glycine, and arginine) and biosynthesized Fe-amino acid nano com- ging from 52.94 ± 1.28% (T3) to 89.60 ± 1.75% (control). It is worth plexes in order to identify their organic species (Table 1). noting that the other constituents of BEOs constantly remained under 10%. According to these results, it can be concluded that the genotype fi 3.2. EO yield & composition used here is de nitely a methyl chavicol chemotype. Given that, the content of the compound showed a decrescent trend by the application BEO yields under the influence of different iron sources were eval- of Fe sources in comparison to control, though constantly remained uated and results are illustrated in Fig. 4. Regardless of fertilizer type, over 50%. fi ff foliar application of Fe contributed to a considerable increase in the EO As mentioned above, we identi ed 72 di erent compounds; the yield in comparison to that of control. EO yield values ranged from highest number of compounds was recorded in T1, T3 and T4 with 72 − − 0.29 ± 0.007 g 100 g 1 in control to 0.53 ± 0.016 g 100 g 1 in n[Fe compounds, followed by T2 with 69 and control with 56 compounds; β β α (His) ], respectively. However, differences between treatments con- Spathulenol, (E)-Nerolidol, (Z)- -Farnesene, -copaene, cis- -berga- 3 α taining Fe nano-complexes (T1, T2 and T3) and Fe-EDDHA motene, (E)-methyl cinnamate, neryl acetate, citronellyl acetate, - − δ (0.39 ± 0.009 g 100 g 1) were significant (P<0.05). The highest EO terpinyl acetate, -Elemene, methyl geranate, (Z)-methyl cinnamate, − yield was recorded in T3 (0.53 ± 0.016 g 100 g 1), followed by T1 carvacrol, thymol, chavicol and neral were those 16 compounds not − − fi (0.51 ± 0.014 g 100 g 1) and T2 (0.43 ± 0.017 g 100 g 1); no sig- identi ed in control, of which 10 were monoterpene derivatives, 5 were nificant difference was observed between T3 and T1. Although the sesquiterpene, and the remaining one was the phenylpropene chavicol. lowest value among Fe-containing fertilizers was achieved in T4 In T3, two sesquiterpenes (Elemol and Spathulenol) and one mono- − (0.39 ± 0.009 g 100 g 1), the yield was significantly higher than that terpene ester (Neryl acetate) were missing. − fi ff of control (0.29 ± 0.007 g 100 g 1). In general, the compounds identi ed belonged to di erent classes of The impact of Fe fertilizers (e.g. Tavallali, 2018), and Fe nano- (a) terpenes including monoterpenes (15 compounds), oxygenated complexes (e.g. Amuamuha et al., 2012; Tavallali, 2018; Yousefzadeh monoterpenes (22 compounds), sesquiterpenes (18 compounds) and and Sabaghnia, 2016) on EO yield in some EO bearing crops have been oxygenated sesquiterpenes (8 compounds), and (b) either non-terpene well documented. In line with previous studies, we found evidence for hydrocarbons or their derivatives (4 compounds), and (c) phenylpro- the importance of the Fe source on EO yield in O. basilicum. On the panoids (5 compounds), of which the latter class boasted the highest other hand, the kind of the amino acid present in the iron nano-complex contribution percentage of the total oils (from 53.93% to 92.24%). fi fertilizers also seemed to be a determinant factor. Fe-amino acid nano- The major constituents (over 1%) of EOs in control were speci ed complexes (T1, T2, T3) were more effective in enhancing the sesqui- by a great proportion of the phenylpropanoid methyl chavicol (89.60%) terpene content of BEO than Fe-EDDHA (T4). Apparently, the amino followed by methyl eugenol (2.57%), n-decane (1.41%), 1,8-cineole acids attached to Fe might have a part to play in modifying the con- (1.06%). Other than methyl chavicol, the major constituents ( > 2%) stituents of BEO. The effects of amino acids on the quality and quantity in the remaining EO samples were as follows: of EO in e.g. Rosmarinus officinalis, hortensis and Matricaria 1,8-cineole (9.4, 9.4 and 12%), linalool (2.30%) and (E)-car- recutita (Foroutan nia et al., 2016; Mehrabi et al., 2013; Omer et al., yophyllene (2.01%) were the main compounds found in the plants re- 2013; Gamal El-Din and Abd El-Wahed, 2005) have been reported. The ceiving T1, respectively. The main volatile components of the plants γ decisive role of T3 and T2 on BEO, to some extent, could be attributed receiving T2 were (E)-caryophyllene (6.13 ± 0.07%), (E)- -bisabolene α to the stimulating action of the amino acids histidine and arginine (3.75 ± 0.04%), -humulene (2.60 ± 0.15%), germacrene D α (Ortiz-Lopez et al., 2000). Histidine and arginine play a vital role in (2.44 ± 0.03%) and trans- -bergamotene (2.27 ± 0.05%), respec- γ plant growth and reproduction, chelation and transport of metal ions, tively. (E)-caryophyllene (9.24 ± 0.08%), (E)- -bisabolene α and biosynthesis regulation of other amino acids (Stepansky and (7.33 ± 0.3%), linalool (5.30 ± 0.06%), -humulene α Leustek, 2006; Abdul-Qados, 2009). (4.79 ± 0.23%), germacrene D (4.52 ± 0.25%) and trans- -berga- The composition of BEOs was analyzed and qualitative and motene (2.80 ± 0.02%) were the main components in the EOs treated

448 V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454

Fig. 3. EDX spectra of (a) Fe-Histidine (n[Fe(His)3]), (b) Fe-Glycine (n[Fe(Gly)3]) and (c) Fe-Arginine (n[Fe(Arg)3]) nano-complexes. by T3, while the major constituents of plants receiving T4 were 1,8- of sesquiterpenes (e.g. germacrene, α-bergamotene, β-farnesene, α- cineole (2.88 ± 0.4%), (E)-caryophyllene (2.53 ± 0.05%) and trans- humulene, γ-bisabolene, and caryophyllene) in EO samples (Table 2), α-bergamotene (2.20 ± 0.07%). the greatest quantity of which was recorded in response to T3. According to the results, foliar application of Fe sources led to a While biosynthesis of EO in plants has genetic determination, en- significant decrease in the content of phenylpropanoids in EO samples vironmental conditions have also been evidenced to play a significant (p < 0.05), notably methyl eugenol and methyl chavicol. As opposed part. Growing conditions (e.g. cultivation method, fertilization, irriga- to monoterpenes, the application of iron sources boosted the proportion tion) which largely determine raw material yield, also determine raw

449 V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454

Table 1 type of antioxidant activity measurement to consider various mechan- − FT-IR bands (cm 1) of amino acids (histidine, glycine, and arginine) and Fe- isms of antioxidant activity (Song et al., 2010). In this study, the anti- amino acid nano-complexes (KBr disk). oxidant activity of BEO in response to different iron sources was eval- uated in a series of in vitro tests. Four antioxidant assays i.e. DPPH, NO, ν(C–O) ν(C]O) ν(NH2) δ(C]O) δ(NH2) H2O2 and TBARS scavenging assays were carried out for determining Histidine 1475 1588 3035 1100,500 1100,500 the antioxidant capacity of EO samples with reference to the con- Arginine 1477 1585 3011, 3195 500,1100 1763 centrations providing 50% inhibition (the IC value) (Table 3). The Glycine 1389 1622 2044, 3225 559 1581 50 antioxidant capacity of a given compound is inversely related to its n[Fe(His)3] 1395 1560 3519 1092 – n[Fe(Gly)3] 1408 1651 3379, 3564 804 – IC50; the lower IC50 of a certain chemical or compound, the greater – n[Fe(Arg)3] 1411 1702 3201, 3262 682 antioxidant activity of the compound (Do et al., 2014). DPPH radical scavenging activity of BEOs are shown in Table 3.In The ultrasound-assisted reaction of Fe(NO ) and His, Gly, or Arg led to the 3 3 all samples, the order of the scavenging activity was as follows: Fe synthesis of iron-amino acid nano-complexes. FT-IR bands of free amino acids nano-complexes > Fe-EDDHA > control. The addition of EO to DPPH and nano complexes both displayed an absorption template in the region of −1 solution induced a rapid decrease in the optical density at 517 nm, and 400–4000 cm . In nano-complexes, ν(C–O), ν(C]O), ν(NH2), δ(C]O) and resulted in the significantly better free radical scavenging activity δ(NH2) were the leading vibrations. Compared to the free amino acids, the NH2 vibrational bands in nano-complexes are very broad and delicate and move (lower IC50 value) about 1.5-4-fold, as compared to control − −1 toward higher frequencies about 3200–3600 cm 1. The absorption bands for (3.89 ± 0.5 mg mL ). − C]O are around 1550-1630 cm 1 in amino acids, which is transferred to In this assay, the plants receiving T3 were the most active samples, higher spectra in nano-complexes. whereas control showed the least activity. Furthermore, all EOs ob- tained by using nano-complexes (T3: 1.02 ± 0.09; T1: 1.35 ± 0.08; − material quality. In chemical terms, EOs are very diverse compounds, T2: 1.63 ± 0.2, respectively) mg mL 1 gave stronger radical scaven- − hence their biosynthesis in the plant occurs along different metabolic ging capacity than those receiving T4 (2.36 ± 0.4 mg mL 1). In the pathways, which are differently modified by the presence and avail- other three antioxidant assays, the same order of efficacy for all samples ability of nutrients in the surrounding environment (Nurzyńska- was recorded: Wierdak, 2013). It may be concluded that Fe sources caused the ter- The ability of BEO treated by different Fe sources to scavenge hy- penoid synthesis pathways in O. basilicum to be transferred from phe- drogen peroxide (H2O2) was determined (Table 3). The effectivity of nylpropanoids and monoterpenes to sesquiterpenes. The positive in- the plants receiving nano-complexes (T3 (2.21 ± 0.3 mg mL-1), T1 fluence of Fe NPs on certain sesquiterpenes in e.g. piperita, (2.55 ± 0.2 mg mL-1), T2 (3.33 ± 0.3 mg mL-1), respectively) to

Carum copticum, Ocimum basilicum and Satureja hortensis (Askary et al., scavenge H2O2 was significantly higher than that observed in T4 2016; Abdossi and Kazemi, 2015; Zahedifar and Najafian, 2015) has (3.77 ± 0.4 mg mL-1) and control (5.52 ± 0.5 mg mL-1). also been reported. TBARS radical scavenging activity of BEOs are illustrated in Table 3. While those plants treated by T3 showed a nonsignificant superiority 3.3. Antioxidant activity (3.22 ± 0.2 mg mL-1) over other Fe nano-complex treatments (T1: 3.51 ± 0.2 mg mL-1; T2: 3.88 ± 0.3 mg mL-1), their TBARS scaven- Oxidation is known to have negative effects, especially in the in- ging activity was markedly higher in comparison to T4 (4.57 ± 0.3 mg ff dustrial context. Oxidation reactions occur in foods and cosmetics, mL-1), and control (6.79 ± 0.6 mg mL-1). The di erence in the ac- fi often because of prolonged exposure to oxygen. Food preservation re- tivity of the latter treatments was also signi cant. presents an issue of concern and requires assurance of protection from NO radical scavenging activity of BEOs are shown in Table 3. The microbial spoilage and prolongation of shelf life (Mimica-Dukić et al., plants supplied with T3 were the most potent NO scavengers 2003). The antioxidants from natural sources could be the alternative to (1.62 ± 0.2 mg mL-1) in comparison to other treatments. However, T1 the problematic synthetic antioxidants in counteracting the oxidative & T2, showed stronger scavenging activity compared to T4 stress associated diseases. (2.99 ± 0.08 mg mL-1). In this assay, control had a minimal scaven- The antioxidant capacities of natural sources cannot be fully de- ging activity, as it removed the nitrite radical at the highest con- scribed with only one method; it is necessary to perform more than one centration (4.89 ± 0.4 mg mL-1).

Fig. 4. Basil (Ocimum basilicum) Essential oil yield affected by different Fe sources. Note: Different letters are significantly different at p < 0.05.

450 V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454

Table 2 Effect of different Fe sources on chemical composition of basil essential oils.

No. Compound Retention index n[Fe(Arg)3] n[Fe(Gly)2] n[Fe(His)3] Fe-EDDHA Control

1 α-Thujene 926 0.004 ± 0.0007d 0.017 ± 0.002b 0.006 ± 0.001c 0.002 ± 0.0006e 0.08 ± 0.008a 2 α-Pinene 933 0.26 ± 0.03a 0.11 ± 0.02bc 0.11 ± 0.01bc 0.15 ± 0.03b 0.08 ± 0.007c 3 Camphene 948 0.02 ± 0.005b 0.02 ± 0.005b 0.01 ± 0.008c 0.02 ± 0.007b 0.10 ± 0.03a 4 Sabinene 972 0.13 ± 0.02a 0.10 ± 0.03bc 0.08 ± 0.006c 0.10 ± 0.03bc 0.11 ± 0.03 ab 5 β-Pinene 976 0.29 ± 0.04a 0.18 ± 0.05c 0.19 ± 0.03c 0.22 ± 0.05b 0.11 ± 0.03d 6 Myrcene 990 0.14 ± 0.05bc 0.15 ± 0.03 ab 0.15 ± 0.02 ab 0.17 ± 0.03a 0.12 ± 0.01c 7 n-Decane 999 0.09 ± 0.007c 0.55 ± 0.02b 0.09 ± 0.009c 0.07 ± 0.01c 1.41 ± 0.13a 8 α-Phellandrene 1005 0.009 ± 0.003a 0.01 ± 0.007a 0.01 ± 0.006a 0.007 ± 0.002b 0.01 ± 0.007a 9 (3Z)-Hexenyl acetate 1006 0.02 ± 0.006a 0.02 ± 0.003a 0.01 ± 0.004b 0.01 ± 0.007b 0.01 ± 0.008b 10 δ-3-Carene 1010 0.001 ± 0.0007c 0.02 ± 0.0008a 0.005 ± 0.001b 0.001 ± 0.0005c 0.02 ± 0.006a 11 α-Terpinene 1016 0.02 ± 0.004a 0.01 ± 0.005b 0.02 ± 0.006a 0.01 ± 0.003b 0.02 ± 0.004a 12 p-Cymene 1023 0.001 ± 0.0005d 0.007 ± 0.0006b 0.004 ± 0.0007c 0.003 ± 0.0006cd 0.01 ± 0.005a 13 Limonene 1027 0.31 ± 0.06 ab 0.28 ± 0.04bc 0.34 ± 0.05a 0.25 ± 0.04c 0.29 ± 0.05b 14 1,8-Cineole 1029 2.49 ± 0.2 ab 2.16 ± 0.2bc 1.99 ± 0.3c 2.88 ± 0.4a 1.06 ± 0.12d 15 (Z)-β-Ocimene 1035 0.012 ± 0.007d 0.031 ± 0.003b 0.024 ± 0.006c 0.011 ± 0.005d 0.04 ± 0.004a 16 Benzene acetaldehyde 1041 0.03 ± 0.004bc 0.02 ± 0.003c 0.05 ± 0.004b 0.7 ± 0.06a 0.02 ± 0.005c 17 (E)-β-Ocimene 1045 0.28 ± 0.07c 0.48 ± 0.06a 0.45 ± 0.06 ab 0.34 ± 0.05bc 0.02 ± 0.009d 18 γ-Terpinene 1056 0.03 ± 0.007b 0.03 ± 0.005b 0.05 ± 0.006a 0.03 ± 0.007b 0.03 ± 0.008b 19 cis-Sabinene hydrate 1065 0.05 ± 0.006b 0.04 ± 0.004bc 0.10 ± 0.03a 0.05 ± 0.005b 0.03 ± 0.007c 20 Fenchone 1087 0.40 ± 0.03b 0.40 ± 0.02b 0.70 ± 0.03a 0.67 ± 0.08a 0.46 ± 0.07b 21 Linalool 1099 2.30 ± 0.13b 1.01 ± 0.03c 5.30 ± 0.06a 1.14 ± 0.06c 0.30 ± 0.07d 22 n-Nonanal 1103 0.02 ± 0.004b 0.02 ± 0.004b 0.02 ± 0.003b 0.01 ± 0.005c 0.05 ± 0.008a 23 endo-Fenchol 1112 0.07 ± 0.006c 0.16 ± 0.04b 0.22 ± 0.05a 0.05 ± 0.004c 0.20 ± 0.07 ab 24 Cis-p-Menth-2-en-l-ol 1120 0.01 ± 0.007c 0.02 ± 0.003bc 0.01 ± 0.004c 0.004 ± 0.0006d 0.03 ± 0.005a 25 Camphor 1142 0.04 ± 0.005c 0.25 ± 0.05a 0.18 ± 0.03b 0.17 ± 0.04b 0.03 ± 0.004c 26 δ-Terpineol 1165 0.05 ± 0.005b 0.04 ± 0.004b 0.12 ± 0.03a 0.03 ± 0.006b 0.01 ± 0.005c 27 Terpinen-4-ol 1175 0.04 ± 0.004c 0.04 ± 0.004c 0.16 ± 0.005a 0.07 ± 0.005b 0.02 ± 0.003d 28 α-Terpineol 1190 0.26 ± 0.03a 0.18 ± 0.03c 0.27 ± 0.03a 0.22 ± 0.02b 0.08 ± 0.005d 29 Methyl chavicol 1196 79.05 ± 1.34c 74.22 ± 1.42c 52.94 ± 1.28d 82.14 ± 1.55b 89.60 ± 1.75a 30 endo-Fenchyl acetate 1219 0.08 ± 0.005b 0.09 ± 0.003b 0.17 ± 0.04a 0.15 ± 0.04a 0.09 ± 0.007b 31 Nerol 1226 0.02 ± 0.006c 0.07 ± 0.005b 0.03 ± 0.004c 0.006 ± 0.0005d 0.12 ± 0.06a 32 Neral 1239 1.37 ± 0.05a 0.008 ± 0.0005d 1.16 ± 0.07b 0.02 ± 0.004c n.d. 33 Chavicol 1251 0.02 ± 0.007a 0.02 ± 0.007a 0.005 ± 0.0006b 0.002 ± 0.0006b n.d. 34 Geranial 1269 1.81 ± 0.06a 0.02 ± 0.006c 1.56 ± 0.08b 0.03 ± 0.005c 0.003 ± 0.0006d 35 (E)-Anethole 1284 0.19 ± 0.07b 0.30 ± 0.06d 0.20 ± 0.07b 0.27 ± 0.04a 0.07 ± 0.006c 36 Thymol 1291 0.002 ± 0.0009b 0.004 ± 0.001a 0.005 ± 0.0009a 0.001 ± 0.0005 n.d. 37 Carvacrol 1298 0.003 ± 0.0008 ab 0.002 ± 0.001bc 0.004 ± 0.001a 0.001 ± 0.0004c n.d. 38 (Z)-Methyl cinnamate 1304 0.001 ± 0.0007c 0.007 ± 0.0008a 0.005 ± 0.0006b 0.002 ± 0.0007c n.d. 39 Methyl geranate 1323 0.003 ± 0.0009c 0.001 ± 0.0006d 0.03 ± 0.006a 0.006 ± 0.0008b n.d. 40 δ-Elemene 1335 0.009 ± 0.0005b 0.002 ± 0.0007c 0.03 ± 0.004a 0.001 ± 0.0007c n.d. 41 α-Terpinyl acetate 1347 0.008 ± 0.0006b 0.004 ± 0.0008c 0.02 ± 0.005a 0.005 ± 0.0006c n.d. 42 Citronellyl acetate 1352 0.006 ± 0.0007c 0.001 ± 0.0005d 0.02 ± 0.006a 0.01 ± 0.006b n.d. 43 Eugenol 1355 0.02 ± 0.004c 0.02 ± 0.007c 0.10 ± 0.08b 0.30 ± 0.06a 0.008 ± 0.0006d 44 Neryl acetate 1363 0.009 ± 0.0005b n.d. 0.04 ± 0.005a 0.009 ± 0.0004b n.d. 45 α-Copaene 1373 0.03 ± 0.009b 0.009 ± 0.0007c 0.06 ± 0.006a 0.004 ± 0.0008d 0.004 ± 0.0007d 46 (E)-Methyl cinnamate 1381 0.04 ± 0.006b 0.01 ± 0.006c 0.12 ± 0.04a 0.04 ± 0.007b n.d. 47 β-Elemene 1390 0.12 ± 0.05b 0.11 ± 0.06b 0.41 ± 0.06a 0.04 ± 0.004c 0.007 ± 0.001d 48 (Z)-Jasmone 1397 0.02 ± 0.004c 0.06 ± 0.006b 0.03 ± 0.005c 0.009 ± 0.0006d 0.08 ± 0.005a 49 Methyl eugenol 1404 0.50 ± 0.05c 0.38 ± 0.4d 0.69 ± 0.3b 0.47 ± 0.05c 2.57 ± 0.07a 50 Cis-α-Bergamotene 1413 0.01 ± 0.006b 0.008 ± 0.0007c 0.03 ± 0.005a 0.003 ± 0.0007d n.d. 51 (E)-Caryophyllene 1416 2.02 ± 0.02c 6.13 ± 0.07b 9.24 ± 0.08a 2.53 ± 0.05c 0.73 ± 0.08d 52 β-Copaene 1426 0.01 ± 0.006a 0.003 ± 0.0008b 0.02 ± 0.006a 0.002 ± 0.0009b n.d. 53 trans-α-Bergamotene 1433 1.42 ± 0.04c 2.27 ± 0.05b 2.80 ± 0.02a 2.20 ± 0.07b 0.06 ± 0.01d 54 (Z)-β-Farnesene 1441 0.05 ± 0.003b 0.02 ± 0.004c 0.08 ± 0.006a 0.006 ± 0.0007d n.d. 55 α-Humulene 1450 0.98 ± 0.19d 2.60 ± 0.15b 4.79 ± 0.23a 1.33 ± 0.14c 0.17 ± 0.07e 56 (E)-β-Farnesene 1455 0.44 ± 0.05b 0.16 ± 0.08c 0.60 ± 0.04a 0.06 ± 0.008d 0.07 ± 0.006d 57 Allo-Aromadendrene 1460 0.02 ± 0.009b 0.02 ± 0.007b 0.04 ± 0.007a 0.006 ± 0.0007c 0.01 ± 0.006b 58 Germacrene D 1478 1.10 ± 0.22c 2.44 ± 0.03b 4.52 ± 0.25a 1.17 ± 0.04e 0.28 ± 0.08d 59 β-Selinene 1482 0.14 ± 0.07a 0.08 ± 0.006b 0.20 ± 0.08a 0.03 ± 0.005bc 0.01 ± 0.005c 60 α-Zingiberene 1493 0.31 ± 0.07b 0.13 ± 0.05c 0.52 ± 0.09a 0.05 ± 0.005d 0.03 ± 0.006d 61 β-Bisabolene 1506 0.09 ± 0.006b 0.03 ± 0.009c 0.14 ± 0.05a 0.01 ± 0.006d 0.007 ± 0.001e 62 γ-Cadinene 1511 0.13 ± 0.02b 0.11 ± 0.03b 0.19 ± 0.04a 0.04 ± 0.005d 0.09 ± 0.005c 63 δ-Cadinene 1521 0.05 ± 0.006b 0.02 ± 0.005c 0.10 ± 0.05a 0.006 ± 0.0008d 0.006 ± 0.0006d 64 (E)-γ-Bisabolene 1537 1.74 ± 0.06c 3.75 ± 0.04b 7.33 ± 0.3a 1.28 ± 0.04c 0.35 ± 0.04d 65 Elemol 1546 0.004 ± 0.0005c n.d. 0.02 ± 0.007a 0.001 ± 0.0006d 0.007 ± 0.0006b 66 (E)-Nerolidol 1561 0.06 ± 0.007a 0.02 ± 0.006b 0.07 ± 0.006a 0.01 ± 0.006b n.d. 67 Spathulenol 1577 0.01 ± 0.004b n.d. 0.02 ± 0.006a 0.001 ± 0.0005c n.d. 68 Caryophyllene oxide 1579 0.04 ± 0.005b 0.01 ± 0.006c 0.07 ± 0.005a 0.006 ± 0.0005d 0.005 ± 0.0008d 69 epi-α-Cadinol 1638 0.46 ± 0.05b 0.37 ± 0.02c 0.50 ± 0.09b 0.23 ± 0.07d 0.60 ± 0.08a 70 β-Eudesmol 1646 0.01 ± 0.007b 0.009 ± 0.0007c 0.05 ± 0.006a 0.01 ± 0.006b 0.007 ± 0.0007c 71 α-Cadinol 1651 0.05 ± 0.006b 0.06 ± 0.007 ab 0.06 ± 0.007 ab 0.01 ± 0.005c 0.08 ± 0.006a 72 α-Bisabolol 1681 0.11 ± 0.06a 0.03 ± 0.005b 0.13 ± 0.07a 0.02 ± 0.004b 0.02 ± 0.005b

Data are mean ± standard deviation of eight replications (pots). Note: Different letters are significantly different at p < 0.05.

451 V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454

Table 3 Radical scavenging activity of basil essential oils in response to different Fe sources.

Properties Fe sources (0.2% w/v)

n[Fe(Arg)3] n[Fe(Gly)3] n[Fe(His)3] Fe-EDDHA Control

DPPH 1.35 ± 0.08b 1.63 ± 0.2b 1.02 ± 0.09a 2.36 ± 0.4c 3.89 ± 0.5d

H2O2 2.55 ± 0.2a 3.33 ± 0.3b 2.21 ± 0.3a 3.77 ± 0.4c 5.52 ± 0.5d TBARS 3.51 ± 0.2b 3.88 ± 0.3c 3.22 ± 0.2a 4.57 ± 0.3d 6.79 ± 0.6e NO 1.89 ± 0.08b 2.07 ± 0.3c 1.62 ± 0.2a 2.99 ± 0.08d 4.89 ± 0.4e

Note: In each row, different letters are significantly different at p < 0.05.

− A large number of naturally occurring substances have been re- 0.111 ± 0.045 mg mL 1). The least activity among Fe fertilizers was cognized to have antioxidant activities, among which phenolics and recorded in T4 (0.200 ± 0.101, 0.181 ± 0.021), which was markedly their derivatives are receiving particular attention (Ibrahim and Jaafar, higher than that in control (0.299 ± 0.112, 0.221 ± 0.102). 2013). A highly positive relationship between total phenols and anti- T3 yielded the most potent EO against 2 g-positive bacteria B. sub- − oxidant activity has been found in many plant taxa (Gülçin et al., 2004). tilis and S. aureus (0.033 ± 0.009, 0.002 ± 0.0007 mg mL 1), and Although the exact mode of action of phenolics has not been well un- two foodborne gram-negative bacteria E. coli and S. typhimurium − derstood, such compounds may exert their effect on biological systems (0.181 ± 0.011, 0.163 ± 0.077 mg mL 1), respectively. Conversely, by different mechanisms including the sequestration of free radicals, the weakest bactericidal activity against both 2 g-positive bacteria B. − hydrogen donation and metallic ion chelation, etc. Moreover, today we subtilis (0.130 ± 0.033 mg mL 1) and S. aureus − know that additive and synergistic effects of phytochemicals in plants (0.072 ± 0.015 mg mL 1), and two foodborne gram-negative bacteria − are responsible for their potent antioxidant activity, not a single com- E. coli (0.833 ± 0.077 mg mL 1) and S. typhimurium − pound (Liu, 2003). BEOs tested here were multi-component mixtures (0.781 ± 0.102 mg mL 1) was recorded in control. T4 showed an in- consisted of a variety of highly functionalized chemical entities, be- termediary activity between Fe nano-complexes and control, in terms of longing to different chemical classes. BEOs contained surprisingly high the ability to disturb bacterial growth (Table 4). MIC of ampicillin and proportions of the phenolic derivative (phenylpropanoid) methyl cha- gentamicin, the reference antibiotic drugs, were determined against − − vicol (~53–90%), and, in minor quantities, other phenylpropanoids gram-positive (B. subtilis: 0.018 mg mL 1; S. aureus: 0.020 mg mL 1) − such as methyl eugenol (~0.4–2.5%), anethole (~0.1–0.3), eugenol and gram-negative bacteria (E. coli: 0.012 mg mL 1; S. typhimurium: − (~0.01–0.3%) and chavicol (~0–0.02%). In addition, two phenolics 0.013 mg mL 1), respectively, and results indicated that in most cases (carvacrol and thymol) and three monoterpene hydrocarbons (α-terpi- both are the most active agents against the pathogens. Exceptions in- nene, γ-terpinene ad sabinene), claimed to be potent antioxidant clude T3 and T1, in which the activity against S. aureus was dramati- agents, also played their parts. cally higher as compared to antibiotics. The activity of BEOs against B.

To sum up, various Fe sources, n[Fe(His)3] in particular, markedly subtilis was very close to the antibiotic. It can be concluded that the boosted the antioxidant activity of O. basilicum (p < 0.05). This is in combination of antibiotics/BEOs administered against the microbial agreement with several other researchers who came to a similar con- strains is likely to augment the antimicrobial activity of the drugs. clusion: Antioxidant activity in Pimpinella anisum, Ocimum basilicum and If a certain drug of plant origin is active in concentrations between − − Portulaca oleracea has been shown to be increased by the application of 0.75 and 1.5 mg mL 1 and 2–2.5 mg mL 1, we assume it as a highly Zn-ALA, Zn-amino acids and Fe-ALA nano-complexes, respectively active and moderately active agent, respectively. The activity beyond (Tavallali et al., 2017, 2018; Tavallali, 2018). The greater influence of n the latter range is considered as weak activity. Taking this into con-

[Fe(His)3] on antioxidant activity of O. basilicum could be attributed to sideration, BEOs were very active against the examined pathogens as antioxidant properties of histidine (Ghasemi et al., 2014; Ortiz-Lopez very low concentrations of oils were sufficient to prevent microbial et al., 2000). Histidine has been known as a potent scavenger of the growth. Although, control groups were very active against the fungi, hydroxyl radical and singlet oxygen (Wade and Tucker, 1998). the plants treated by T3 yielded EOs with 5- and 3.3-fold increase in the activity against C. albicans and A. niger, respectively. This comes to 3.4. Antimicrobial activity mean that C. albicans was considerably more sensitive to the drug than A. niger. ff The minimum inhibitory concentration (MIC) derived from serial All samples including control were very e ective against the bac- − dilutions (0–0.3 mg mL 1) of BEOs against the pathogens was char- teria examined; compared to control, plants receiving T3 showed a ~4- acterized. MIC is the lowest concentration of a drug which prevents fold increase in the activity against B. subtilis, while the sensitivity of S. visible growth of the pathogen(s) in a certain medium. MIC of keto- aureus to this treatment was 36-fold larger than control. The suscept- conazole, a synthetic reference antibiotic drug, against two fungi ibility of 2 g-negative bacteria, E. coli and S. typhimurium, to this − − fi strains, C. albicans (0.008 mg mL 1) and A. niger (0.009 mg mL 1) was treatment was also signi cant and for both pathogens showed about recorded. Although, antifungal activity of the synthetic drug was 4.5-fold increase. It was evident that 2 g-positive bacteria (B. subtilis and markedly higher than that of BEOs, EOs were still highly active against S. aureus) were particularly more sensitive to the action of EOs than the fungi strains. Regardless of the iron sources applied, the viability of gram-negative bacteria. According to Burt (2004), EOs are slightly the pathogens was significantly affected by foliar application of ferti- more active against gram-positive than gram-negative bacteria, since lizers as compared to control. On the other hand, the performance of gram-negative organisms possess an outer membrane surrounding the ff nano-complexes was particularly stronger in comparison to Fe-EDDHA. cell wall, relatively e ective in protecting the organism against anti- Generally, the trend for the activity towards six examined strains of bacterial drugs (Ratledge and Wilkinson, 1988). ff pathogens followed similar order of efficacy (Table 4). The MICs ranged A number of compounds belonging to di erent chemical classes are − from 0.058 to 0.299 mg mL 1, indicating different susceptibility of the recorded in BEOs. Therefore, antimicrobial activity of BEOs is not at- pathogens to BEOs. In detail, the application of T3 presented the lowest tributable to one particular mechanism; it is likely that there are several MIC against two foodborne fungi C. albicans and A. niger targets in the cell (Burt, 2004). Lipophilicity plays an especially vital − (0.058 ± 0.006, 0.066 ± 0.008 mg mL 1), followed by T1 role in antimicrobial activity of EOs, as it enables them to distribute in − (0.087 ± 0.021, 0.071 ± 0.008 mg mL 1) and T2 (0.145 ± 0.031, the lipids of the organism cell membrane and mitochondria. The most

452 V. Tavallali, et al. Plant Physiology and Biochemistry 144 (2019) 445–454

Table 4 Antimicrobial activity of basil essential oils in response to different Fe sources.

− Species MIC (mg mL 1)

n[Fe(Arg)3] n[Fe(Gly)3] n[Fe(His)3] Fe-EDDHA Control

Fungi C. albicans 0.087 ± 0.021a 0.145 ± 0.031b 0.058 ± 0.006a 0.200 ± 0.101c 0.299 ± 0.112c A. niger 0.071 ± 0.008a 0.111 ± 0.045c 0.066 ± 0.008a 0.181 ± 0.021b 0.221 ± 0.102d Bacteria B. subtilis 0.045 ± 0.004a 0.071 ± 0.006b 0.033 ± 0.009a 0.097 ± 0.020c 0.130 ± 0.033d S. aureus 0.008 ± 0.0006a 0.033 ± 0.011b 0.002 ± 0.0007a 0.061 ± 0.009c 0.072 ± 0.015d E. coli 0.309 ± 0.061b 0.411 ± 0.101c 0.181 ± 0.011a 0.582 ± 0.111d 0.833 ± 0.077e S. typhimurium 0.322 ± 0.021b 0.512 ± 0.120c 0.163 ± 0.077a 0.604 ± 0.115c 0.781 ± 0.102d

Values are means of Minimal Inhibitory Concentration (MIC) in eight replications (pots) ± sd. (standard deviation). Note: In each row, different letters represent significant differences among treatments (p < 0.05). important mechanisms are thought to be the disturbance of cytoplasmic antioxidant activity of basil, and to screen out a new source of natural membrane, damage to membrane proteins, leakage of cell contents, and effective drugs against some common strains of foodborne fungi coagulation of cytoplasm, depletion of the proton motive force and and bacteria. The initial results obtained are quite encouraging since most likely preventing enzyme action via binding to proteins (Devi EOs tested exhibited potent antioxidant effect, and antifungal and an- et al., 2010). tibacterial activities. The inherent biological and pharmacological activities of a given oil is related to the chemical configuration of the components, the pro- Declaration of competing interest portions in which they are present and to interactions between them. The EOs possessing the strongest antibacterial properties against food The manuscript has been read and approved by all named authors borne pathogens mainly contain a high percentage of oxygenated and that there are no other persons who satisfied the criteria for au- monoterpenes and phenolic compounds (Aggarwal et al., 2002). BEOs thorship but are not listed. examined here were particularly rich in such compounds, including the phenylpropanoids methyl chavicol (~53–90%), methyl eugenol References (~0.4–2.5%), anethole (~0.1–0.3), eugenol (~0.01–0.3%) and cha- vicol (~0–0.02%). Phenylpropenes, notably eugenol, methyl chavicol Abdossi, V., Kazemi, M., 2015. Effect of nano-iron chelate on chemical composition and and anethole, have a role in plant defense given they show activity antimicrobial properties of Carum copticum L. essential oil and its main terpenes from Iran. Bangladesh J. Bot. 44 (4), 537–542. against a wide range of pathogens (Atkinson, 2016). Additionally, two Abdul-Qados, A.M.S., 2009. Effect of arginine on growth, yield and chemical constituents phenolic compounds of BEOs, carvacrol and its isomer thymol, are well- of wheat grown under salinity condition. Acad. J. Plant Sci. 2, 267–278. known for their strong antimicrobial activity (Memar et al., 2017). Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ α Mass Spectrometry. Allured Publishing, Carol Stream, IL. 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